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Molecular Biology and Genetics
725 N. Wolfe St.
Baltimore MD 21205
Molecular mechanisms of retinal development and disease; the role of Frizzled receptors in mammalian development
Biology: Frizzled receptors in development and disease Our laboratory has focused for the past two decades on a large family of cell-surface receptors called Frizzled. This name refers to the appearance of fruit flies in which the receptor gene is mutated: the hairs and bristles on the body surface of Frizzled mutant flies are oriented inappropriately. In mammals, including humans, there are ten closely related Frizzled genes. In the mid-1990s, we showed, in collaboration with the laboratory of Roel Nusse at Stanford, that the principal ligands for Frizzleds are the Wnt proteins. There are 19 Wnt genes in mammals, and current evidence suggests that each Frizzled can bind to multiple of Wnts and each Wnt can bind to multiple Frizzleds. To add to the complexity, three distinct types of signals can be sent from Frizzled receptors to the cell interior. Our current focus is on defining the roles of Frizzled receptors in mammalian development. The foundation of our approach is the production and analysis of mice carrying targeted null or conditional null mutations in one or more Frizzled genes. We have constructed such lines for each of the ten Frizzleds, as well as for other genes that act in the same signaling pathways. This genetic analysis has revealed both diversity and unity in the functions of different Frizzled receptors, and has revealed the requirement for Frizzled signaling in a wide variety of developmental contexts, including axon guidance, vascular growth and differentiation, inner ear development, neural tube and palate closure, kidney development, and hair orientation on the body surface. In the context of vascular growth and differentiation, we identified a novel ligand (Norrin) that acts exclusively on the Frizzled (Frizzled4) that controls vascular development. In humans, mutations in the Norrin or Frizzled4 genes, or in genes coding for a co-receptor (Lrp5) or chaperone (Tspan12) produce vascular defects similar to their mouse counterparts. Current experiments are aimed at (1) identifying additional roles for Frizzleds in mammalian development, homeostasis, and disease, and (2) elucidating the molecular logic of Frizzled signaling. Technology: new tools for mouse genetics and neuroscience For the past decade we have been developing new genetic tools for visualizing and manipulating single identified cells in mice. Our approaches build on existing methods that use pharmacologic control of cre-loxP recombination. In one set of experiments we created a variety of knock-in alleles in which a pair of loxP sites flank the coding region and 3’ untranslated region (UTR) of a gene of interest, with a reporter gene inserted distal to the 3’-most loxP site. When the conditional knockout allele is placed over a WT allele, cre-mediated recombination creates heterozygous cells that exhibit reporter expression under the control of the promoter for the gene of interest. When the conditional knockout allele is placed over a conventional null allele in the gene of interest, cre-mediated recombination creates mutant cells that are similarly marked by expression of the reporter. By pharmacologic titration of cre activity, one can generate sparse mosaics of recombined cells, thereby permitting an analysis of individual mutant cells in an otherwise WT environment. Genetically-directed sparse recombination is especially useful for determining the morphology of genetically defined neurons. For some extremely large and complex neurons – such as forebrain cholinergic neurons, dopaminergic amacrine cells in the retina, and large cutaneous sensory neurons in the skin – visualizing the full structure of individual axonal or dendritic arbors has required labeling densities of only one or a few neurons per animal. Experiments currently in progress are extending these approaches to the visualization of distinct subcellular structures in individual identified cells and to the tracing of cell lineages in a variety of contexts.
Wu, H., Luo, J, Yu, H., Rattner, A., Mo, A., Smallwood, P.M., Erlanger, B., Wheelan, S.J., and Nathans, J. (2014) Cellular resolution maps of X-chromosome inactivation: implications for neural development, function, and disease. Neuron 81: 103-119.
Chang, H., Wang, Y., Wu, H., and Nathans, J. (2014) Whole mount imaging of mouse skin and its application to the analysis of hair follicle patterning and sensory axon morphology. Journal of Visualized Experiments: e51749.
Hua, Z.L., Jeon, S., Caterina, M., and Nathans, J. (2014) Frizzled3 is required for the development of multiple axon tracts in the mouse central nervous system. Proceedings of the National Academy of Sciences USA 111: E3005-3014.
Wu, H., Williams, J., and Nathans, J. (2014) Complete morphologies of basal forebrain cholinergic neurons in the mouse. eLife 3: e02444.
Zhou, Y., Wang, Y., Tischfield, M., Williams, J., Smallwood, P.M., Rattner, A., Taketo, M.M., and Nathans, J. (2014) Canonical Wnt signaling components in vascular development and barrier formation. Journal of Clinical Investigation 124: 3825-3846.
Hua, Z.L., Chang, H., Wang, Y., Smallwood, P.M., and Nathans, J. (2014) Partial interchangeability of Frizzled3 and Frizzled6 in tissue polarity signaling for epithelial orientation and axon growth and guidance. Development 141: 3944-3954.
Zhou, Y., and Nathans, J. (2014) Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical Wnt signaling. Developmental Cell 31: 248-256.
Rattner, A., Wang, Y., Zhou, Y., Williams, J., and Nathans, J. (2014) The role of the hypoxia response in shaping retinal vascular development in the absence of Norrin/Frizzled4 signaling. Investigative Ophthalmology and Visual Science 55: 8614-8625.
Mo, A., Mukamel, E.A., Davis, F.P., Luo, C., Henry, G.L., Picard, S., Urich, M.A., Nery, J.R., Sejnowski, T.J., Lister, R., Eddy, S.R., Ecker, J.R., and Nathans, J. (2015) Epigenomic signatures of neuronal diversity in the mammalian brain. Neuron 86: 1369-1384.
Chang, H., Cahill, H., Smallwood, P.M., Wang, Y., and Nathans, J. (2015) Identification of Astrotactin2 as a genetic modifier that regulates the global orientation of mammalian hair follicles. PLOS Genetics 11: e1005532.
Chang, H., Smallwood, P.M., Williams, J., and Nathans, J. (2016) The spatio-temporal domains of Frizzled6 action in planar polarity control of hair follicle orientation. Developmental Biology 409: 181-193.
Mo, A., Luo, C., Davis, F.P., Mukamel, E.A., Henry, G.L., Nery, J.R., Urich, M.A., Picard, S., Lister, R., Eddy, S.R., Beer, M.A., Ecker, J.R., and Nathans, J. (2016) Epigenomic landscapes of retinal rods and cones. eLife 5: e11613.
Wang, Y., Williams, J., Rattner, A., Wu, S., Bassuk, A.G., Goffinet, A.M., and Nathans, J. (2016) Patterning of papillae on the mouse tongue: A system for the quantitative assessment of planar cell polarity signaling. Developmental Biology 419: 298-310.
Chang, H., Smallwood, P.M., Williams, J., and Nathans, J. (2017) Intramembrane proteolysis of Astrotactins. Journal of Biological Chemistry 292: 3506-3516.
Cho C., Smallwood P.M., and Nathans J. (2017) Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation. Neuron 95: 1056-1073.
Sabbagh, M.F., Heng, J.S., Luo, C., Castanon, R.G., Nery, J.R., Rattner, A., Goff, L.A., Ecker, J.R., Nathans, J. (2018) Transcriptional and epigenomic landscapes of CNS and non-CNS vascular endothelial cells. eLife 7: e36187.
Peng, X., Emiliani, F., Smallwood, P.M., Rattner, A., Lei, H., Sabbagh, M.F., and Nathans, J. (2018) Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions. eLife 7: e40982.
Wang, Y., Cho, C., Williams, J., Smallwood, P.M., Zhang, C., Junge, H.J., and Nathans, J. (2018) Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood-brain barrier and blood-retina barrier development and maintenance. Proceedings of the National Academy of Sciences USA 15: E11827-E11836.
Wang, Y., Sabbagh, M.F., Gu, X., Rattner, A., Williams, J., and Nathans, J. (2019) Beta-catenin signaling regulates barrier-specific gene expression in circumventricular organ and ocular vasculatures. eLife 8: e43257.
Heng, J.S., Rattner, A., Stein-O’Brien, G.L., Winer, B.L., Jones, B.W., Vernon, H.J., Goff, L.A., and Nathans, J. (2019) Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain studied at single-cell resolution. Proceedings of the National Academy of Sciences USA 116: 9103-9114.
Cho, C., Wang, Y., Smallwood, P.M., Williams, J., and Nathans, J. (2019) Dlg1 activates beta-catenin signaling to regulate retinal angiogenesis and the blood-retina and blood-brain barriers. eLife 8: e45542
Cho, C., Wang, Y., Smallwood, P.M., Williams, J., Nathans, J. (2019) Molecular determinants in Frizzled, Reck, and Wnt7a for ligand-specific signaling in neurovascular development. eLife 8: e47300.
Rattner, A., Williams, J., and Nathans J. (2019) Roles of HIFs and VEGF in angiogenesis in the retina and brain. Journal of Clinical Investigation 130: 126655.